Invited review: decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art

Abstract

Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and noncoding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may coexist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function. Genetic mutations that cause neurodegenerative disorders disrupt healthy gene expression at diverse levels, from chromatin remodelling, transcription, splicing, through to axonal transport and repeat-associated non-ATG (RAN) translation. We address neurodegeneration in repeat expansion disorders [Huntington's disease, spinocerebellar ataxias, C9ORF72-related amyotrophic lateral sclerosis (ALS)] and in diseases caused by deletions or point mutations (spinal muscular atrophy, most subtypes of familial ALS). Some neurodegenerative disorders exhibit broad dysregulation of gene expression with the synthesis of hundreds to thousands of abnormal messenger RNA (mRNA) molecules. However, the number and identity of aberrant mRNAs that are translated into proteins - and how these lead to neurodegeneration - remain unknown. The field of RNA biology research faces the challenge of identifying pathophysiological events of dysregulated gene expression. In conclusion, we discuss current research limitations and future directions to improve our characterization of pathological mechanisms that trigger disease onset and progression.

Keywords: RNA-mediated diseases; RNA/protein toxic gain-of-function; altered gene expression; neurodegeneration; protein loss-of-function.

Figure 1

Neuronal expression of protein-coding genes. Diagram highlighting mRNA biogenesis and processing, nuclear export, axonal transport and mRNA translation. (1) Chromatin remodelling; (2) RNA polymerase II (RNA Pol. II) dependent transcription; (3) co-transcriptional processing: 5′-end capping, splicing/alternative spicing, 3′-end cleavage and poly-adenylation; (4) nuclear export of mRNAs; (5) axonal transport of mRNAs; and (6) translation of mRNAs for the biosynthesis of proteins.

Figure 2

Mechanisms conferring protein loss and toxic gain-of-function effects. The diagram illustrates pathogenic mutations (repeat expansions, deletions, point mutations) that may occur either in noncoding or coding regions of the genome (left and right sides, respectively). (A) Protein loss-of-function. Haploinsufficiency can occur when the level of a particular mRNA is down-regulated due to mutations in noncoding regions of genes such as in promoters/introns, or if the promoter is subjected to histone/DNA modifications (transcriptional repression), but also if mutations in 5′ or 3′ untranslated regions (UTRs) decrease mRNA stability. Protein loss-of-function can also occur when mutations in coding regions alter directly the activity of the mutated protein (misfolding, alteration of the active site). (B) Protein toxic gain-of-functions are caused by mutations in coding regions that either promote abnormal interactions, increase the interaction of the mutated protein with its natural binders and/or promote misfolding/aggregation.

Figure 3

RNA toxic gain-of-function mechanisms. (A) Protein sequestration of RNA-binding proteins that avidly interact with the repeat expanded pre-mRNA/mRNA. (B) Formation of RNA foci. (C) Repeat-associated non-ATG (RNA) translation. (D) RAN translation leads to the formation of repeat-peptide proteins that usually aggregate.

Figure 4

Model for regulation of exon-7 splicing in SMN1 and SMN2. Schematic representation of positive and negative effectors that regulate exon-7 splicing in SMN1 (A) and SMN2 (B) genes. The acronym ASF was used for alternative splicing factors. Exons 7 of SMN1 or SMN2 are represented in boxes that include the DNA sequences of the ESE/ESS motifs. ISS sequences are located in introns flanking exon 7 of SMN2. Arrows represent binding of ASF to the highlighted DNA elements or proteins. Factors that promote or inhibit the inclusion of exon-7 are respectively labelled above or below SMN exons/introns. T lines represent binding inhibition/inhibitory effect of ASF. (A) SRSF1 recognizes a +6 ESE sequence in SMN1 exon-7 promoting inclusion of exon-7. A downstream AG-rich ESE in exon-7 promotes exon-7 inclusion through binding of PSF and hnRNPM , which in turn stimulates the recruitment of the splicing factor U2AF65 to the flanking intron-7. (B) The ESE sequence altered by a C/T transition at position +6 in SMN2 exon-7 was initially suggested to reduce exon-7 splicing because of a decreased interaction with SRSF1 ,,. However, the C/T transition also forms a composite ESS that promotes exon-7 skipping by interaction with the alternative splicing inhibitors hnRNPA1 , and Sam68 . Furthermore, the activities of both hnRNPQ2 and Q3 antagonize the positive exon-7 splicing role of hnRNPQ1 bound to the +6 ESE . Several base changes in SMN2 introns 6 and 7 also promote SMN2 exon-7 exclusion: (i) an ISS Element 1 in intron-6 (−75 to −89) through binding of p33 ; (ii) an ISS-N1 site located in intron-7 (+10 to +24) that provides binding sites for hnRNPA2 and B1 ; (iii) an ISS in intron-7 (A/G transition at position +100) that binds hnRNPA1 and inhibits splicing of exon-7 cooperatively with the binding of the same protein to the exon-7 ESS site . In contrast, SMN2 exon-7 inclusion is promoted via two ESE sites: (i) the composite +6 ESE which provides interaction for hnRNPQ1 ; and (ii) the AG-rich ESE that provides overlapping binding sites for the splicing factors PSF , hnRNPM and hTra2-β1 . The direct interactions of hTra2-β1 with the alternative splicing factors SRp30c , hnRNPG and RBMX/Y increase the splicing activity of ESE-bound hTra2-β1, stimulating in turn exon-7 inclusion. Interestingly, a silent C/G transition identified in AG-rich ESE at position +27 (codon Gly287) in some SMA II or III patients which present mild clinical phenotypes, creates an ESE for SRSF1, which in turn promotes exon-7 splicing and the production of full-length SMN2 mRNAs . However, this transition also disrupts a splicing-inhibitory hnRNPA1 binding site indirectly promoting SMN2 exon-7 inclusion .